Human Immunodeficiency Virus (HIV-1) entry is mediated by the viral envelope glycoprotein (Env). Binding of the gp120 domain of Env to CD4 leads to conformational changes that expose the coreceptor binding site. Interaction with the coreceptor activates the gp41 domain of Env to promote fusion between the viral and cellular membranes. HIV-1 Env remains an attractive target for vaccines and antiviral therapies as it is the only viral protein exposed on the surface of HIV-1 virions. Based on the metastable nature of the unliganded HIV-1 Env trimer, antiviral strategies have followed three main concepts: 1) competitive inhibition without allosteric activation; 2) premature allosteric activation in the absence of target cell membranes; and 3) diversion into off- pathway conformations. This application proposes to test the hypothesis that the stabilization of the ground state conformation of Env, which prevents the activation of Env, is an underutilized antiviral strategy. Support for this hypothesis is based on the impact on Env conformation of antibodies that neutralize up to 98% of all HIV-1 isolates. To test this hypothesis, we have established single-molecule fluorescence resonance energy transfer (smFRET) imaging methods to directly visualize the conformational dynamics of single Env molecules within the native trimer on the surface of intact virions. Fluorophores introduced into variable regions V1, V4, and V5 of gp120 of the NL4-3 strain of HIV-1 allowed the time-resolved monitoring of structural dynamics of individual gp120 domains within the context of the native Env trimer on the surface of intact virions. These data reveal that the unliganded HIV-1 Env is dynamic and intrinsically accesses the receptor- and coreceptor- stabilized conformations. The establishment of smFRET for HIV-1 Env allows insights into the inner workings of this molecular machine and how it is activated for fusion by the two-step receptor and coreceptor mechanism. Moreover, it offers a fast and reliable assay for the conformational state of HIV-1 Env on the surface of virions. Using this approach we have shown that the broadly neutralizing antibodies, VRC01, PG16, PGT128, PGT145, and 2G12, stabilize HIV-1 Env in its ground-state conformation despite engaging Env in fundamentally different ways. VRC01 recognizes an epitope in the CD4-binding site; PG16, PGT128, and PGT145 bind V1/V2 at the apex of the trimer; and 2G12 recognizes conserved glycans at the base of the V3 loop. These results suggest that stabilization of the ground state but not of an activated intermediate or off- pathway conformation represents a powerful antiviral strategy. Notably, the above experiments were performed with the neutralization-sensitive NL4-3 HIV-1 isolate. Here, we will test whether broadly neutralizing antibodies similarly function on Env from clinical HIV-1 isolates. We will also test the conformational consequences of entry inhibitors, such as BMS-626529 that may similarly stabilize the ground state. In so doing, the proposed studies have the potential to establish that ground state stabilization is an effective way to antagonize viral membrane fusion machines.